Macrophages are relatively large (10-20 um), actively motile phagocytic cells that develop from blood-borne monocytes, which in turn originate in the bone marrow. Upon activation, macrophages have been observed to undergo several functional, biochemical and morphological changes, including membrane ruffling, peroxide elaboration, increased expression of Ia antigens and increased secretion of plasminogen. Also, macrophages are considered to be a key element of the immune system. When activated they have been associated with destroying foreign particles and decrepit cells, and also more recently have been identified as providing resistance to and/or eradication of neoplastic disease. The development of activated macrophages and precursor monocytes which display these activities requires the simultaneous presence of effective activation signals and receptive mononuculear phagocytes.
It has been generally accepted that activation of macrophages can be induced by certain lymphokines, referred to as macrophage activating factors or MAF. Cameron et al., J. Clin. Invest. 63: 977 (1979); Mantavani et al., Int. S. Cancer, 25: 691 (1980); and, Kleinerman et al., J. Clin. Invest. 72: 304 (1983).
It is unknown whether MAF is a distint lymphokine or alternatively is in whole or in part composed of other lymphokines. Some researchers have suggested that in the murine system, MAF is composed of gamma interferon ("IFN-.gamma."). See Roberts and Vasil, J. Interferon Res., 2: 519 (1982); and, Svedersky et al., J. Exp. Med., 159: 812 (1984).
However, lymphokines other than IFN-.gamma. also are thought possibly to activate macrophages to the point where they are capable of killing tumor cells. Macrophages produce the lymphokine interleukin 1 ("IL-1") which is known to stimulate the growth of skin cells, assist in the healing of wounds and cause inflammation and fever. Onozaki et al., J. Immunol., 135: 314 (1985), recently have suggested that IL-1 also promotes tumoricidal activity of fresh monocytes.
It has been reported that macrophages and precursor monocytes also may be activated to display tumoricidal activity by treatment with various reagents, such as the bacterial products lipopolysaccharide ("LPS"), Sone et al., Cancer Res., 42: 2227 (1982), or peptidoglycan or analogs thereof, such as muramyl dipeptide ("MDP"), Nagao et al., Infec. Immun., 24: 304 (1979), Kleiner et al., Cancer Res., 43: 2010 (1983). Other macrophage activating reagents include the tumor promotor phorbol myristate acetate ("PMA"), Pick et al., Ann. N.Y. Acad. Sci., 332: 378 (1979), and ionophores, Pick et al., supra, and Hund et al., Nature (London), 265: 543 (1979).
Recent research reports have indicated that effective activation of macrophages to display nonspecific tumoricidal activity is not the result of a single signal but requires a series of reactions. Meltzer in "Lymphokines," Pick and Landy, eds., Academic Press, New York, pp. 319-343 (1981), postulates that the activation of macrophages to display tumoricidal activity requires the recruitment or accumulation of blood monocytes at a reaction site, such as at an infection, whereat the monocytes differentiate into competent mononuclear phagocytes. After this initial phase of monocyte differentiation, the monocytes are primed into a receptive state by an initial signal derived from a lymphokine. Final maturation of the primed macrophage into cytotoxic activity requires triggering via a second signal derived, for instance, from LPS. When employed in tandem, the concentrations of the priming signal (lymphokine) and triggering signal (LPS) required for a particular level of macrophage activation are much lower than if these signals are used alone, indicating a synergistic cooperation between such signals.
A relationship similar to the lymphokine-LPS cooperation has been reported for LPS and IFN-.gamma.. Schultz, J. Interferon Res., 2: 459 (1982). The development of recombinant DNA technology combined with in vitro cellular bioassays has lead to the cloning of the cDNAs of IFN-.gamma., IL-1 and other lymphokines. The availability of highly pure, recombinant-derived IFN-.gamma. has confirmed previous reports of its MAF activity and the requirement of a second triggering signal, such as LPS, to effectively induce IFN-.gamma. to display non-specific tumoricidal activity.
The present invention concerns the use of GM-CSF to stimulate macrophages and precursor monocytes to mediate nonspecific tumoricidal activity, which activation is not dependent upon the presence of a costimulator, such as LPS or IFN-.gamma.. GM-CSF is a particular type of colony stimulating factor ("CSF"). CSF refers to a family of lymphokines that induce progenitor cells found in the bone marrow to differentiate into specific types of mature blood cells. The particular type of mature blood cell that results from a progenitor cell depends upon the type of CSF(s) present. For instance, erythropoietin is believed to cause progenitor cells to mature into erythrocytes while thrombopoietin is thought to drive progenitor cells along the thrombocytic pathway. Similarly, granulocyte-macrophage colony formation is dependent on the presence of GM-CSF. Although the ability of CSF, including GM-CSF, to induce the maturation and proliferation of white cells from bone marrow progenitors is well known, heretofore the capacity of GM-CSF to singularly activate macrophages or precursor monocytes to mediate nonspecific tumoricidal activity has been unknown.